Note: Descriptions are shown in the official language in which they were submitted.
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Gluconobacter suboxydans Sorbitol Dehydrogenase,
Genes and Methods of Use Thereof
Background of the Invention
Field of the Invention
The invention relates to the fields of molecular biology, bacteriology and
industrial fermentation. More specifically, the invention relates to the
identification and isolation of nucleic acid sequences and proteins for
subunits of
a novel, membrane bound sorbitol dehydrogenase in Gluconobacter suboxydans.
The invention further relates to the fermentative production of L-sorbose from
D
sorbitol and the subsequent production of 2-keto-L-gulonic acid.
Related Art
Sorbitol dehydrogenase (SDH) is an enzyme responsible for the efficient
conversion of D-sorbitol into L-sorbose during sorbose fermentation in the
process of the manufacturing of 2-keto-L-gulonic acid (2-KLG), an important
precursor for vitamin C synthesis. Gluconobacter possesses several SDHs, which
may be categorized according to their cofactor requirement: ( 1 ) NAD-
dependent,
(2) NADP-dependent and (3) NAD(P)-independent types. Among them,
NAD(P)-independent enzyme is believed to be directly involved in efficient
production of sorbose during industrial sorbose fermentation (Cummins, J. T.
et
al., J. Biol. Chem., 224, 323; 226, 3 O1 (1957)).
The process of manufacturing L-sorbose from D-sorbitol is typically
performed by fermentation with an acetic acid bacterium such as Gluconobacter
suboxydans and Acetobacter xylinum. At room temperature, 96-99% of
conversion is made in less than 24 hours (Liebster, J. et al., Chem. List.,
50:395
(1956)).
CONFfRMATION COPY
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L-sorbose produced by the action of SDH is a substrate in the production
of 2-keto-L-gulonic acid (2-KLG). A variety of processes for the production of
2KLG are known. For example. the fermentative production of 2-KLG via
oxidation of L-sorbose to 2-KLG via a sorbosone intermediate is described for
processes utilizing a wide range of bacteria: Gluconobacter oxydans (U.S. Pat.
Nos. 4,935,359; 4,960,695; 5,312.741; and 5,541,108); Pseudogluconobacter
saccharoketogenes (U.S. Pat. No. 4.877.735; European Pat. No. 221 707);
Pseudomonas sorbosoxidans (U.S. Pat. Nos. 4,933,289 and 4,892,823); mixtures
of microorganisms from these and other genera, such as Acetobacter, Bacillus,
Serratia, Mycobacterium, and Streptom~~ces (U.S. Pat. Nos. 3,912,592;
3,907,639;
and 3,234,105); and novel bacterial strains (U.S. Pat. No. 5,834,231).
A number of enzymes involved in the fermentative oxidation of sorbitol,
sorbose and sorbosone are identified in the literature. U.S. Patent Nos.
5,88,786;
5,861,292; 5,834,263 and 5,753,481 disclose nucleic acid molecules encoding
and/or isolated proteins for L-sorbose dehydrogenase and L-sorbosone
dehydrogenase; and U.S. Patent No. 5,747,301 discloses an enzyme with
specificity for sorbitol dehydrogenase.
In addition to distinguishing Gluconobacter SDH's on the basis of
cofactor requirements, other physical characteristics may be found in the
literature
that distinguish these different enzymes. For example, the sorbitol
dehydrogenase
identified in U. S. Patent No. 5,747,301 is distinguished on the basis of
subcellular
location (membrane-bound) and a haloenzyme molecular weight of 800 ~ 50 kDa
(10 homologous subunits of 79 ~ 5 kDa). The membrane-bound D-sorbitol
dehydrogenase isolated by Shinagawa et al.(E. Shinagawa, K. Matsushita, O.
Adachi and M. Ameyama (Agric. Biol. Chem., 46, 135-141, 1982)) consisted of
three kinds of subunits with molecular weights of 63,000, 51,000 and 17,000.
In an effort to improve the productivity of commercial fermentation in the
production of 2 KLG, the inventors have identified a novel, membrane-bound
sorbital dehydrogenase in a strain of G. suboxydans that is distinct from
others
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described in the literature (Choi, E. S. et al., FEMS Microbiol. Lett., 125:45
( 1995)).
Summary of the Invention
This invention pertains to a novel, membrane-bound sorbitol
dehydrogenase of Gluconobacter suboxydans. The isolated sorbitol
dehydrogenase enzyme comprises three subunits: a first subunit of 75 kDa
containing pyrroloquinoline quinone (PQQ) as cofactor; a second subunit of 50
kDa being a cytochrome c; and a third subunit of 29 kDa playing a very
important
role in the stability and the catalytic activity of the enzyme.
The present invention provides nucleic acid molecules for the 3 protein
subunits of the Gluconobacter sorbitol dehydrogenase described herein. In a
first
specific embodiment, the invention provides an isolated nucleic acid molecule
drawn to the first SDH subunit (75 kDA) identified by SEQ ID NO:1. In a second
specific embodiment, the invention provides an isolated nucleic acid molecule
drawn to the second SDH subunit (50 kDA) identified by SEQ ID N0:2. In a
third specific embodiment the invention provides an isolated nucleic acid
molecule drawn to the third SDH subunit (29 kDA) identified by SEQ ID N0:3.
Other related embodiments are drawn to vectors, processes for producing the
same and host cells carrying said vectors.
The invention also provides isolated nucleic acid molecules encoding the
three subunits of the SDH of the invention. In one specific embodiment, the
invention provides a cloned nucleic acid molecule encoding the 75 kDa and 50
kDa subunits. The structural genes for the first and the second subunit of
sorbitol
dehydrogenase are 2,265 by and 1,437 bp, respectively, in size and are
clustered
in the cloned nucleic acid molecule which is a 5.7 kb Pstl DNA fragment that
defines the operon. In another specific embodiment, the invention provides a
cloned nucleic acid molecule encoding the third, 29 kDa, SDH subunit protein.
The structural gene coding for the third subunit is 921 by in size and found
in a
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4.5 kb Clal DNA fragment. Other related embodiments are drawn to vectors,
processes to make the same and host cells containing said vectors.
The invention is also drawn to isolated polypeptides for the three subunits
of the SDH described herein.
The invention also provides a method for the production of D-sorbose
comprising: (a) transforming a host cell with at least one isolated nucleotide
sequence selected from the group consisting of a polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:1; a polynucleotide comprising the
polynucleotide sequence of SEQ ID N0:2; and a polynucleotide comprising the
polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating
said
transformed host cell.
Another aspect of the invention is drawn to a method for production of 2-
KLG comprising: (a) transforming a host cell with at least one isolated
nucleotide
sequence selected from the group consisting of a polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:1; a polynucleotide comprising the
polynucleotide sequence of SEQ ID N0:2; and a polynucleotide comprising the
polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating
said
transformed host cell.
Brief Description of the Figures
Figure 1 presents DEAE-TSK column chromatography using a sodium
acetate buffer of pH 5.0 (A) and pH 6.0 (B).
Figure 2 presents SDS-PAGE analysis of peak I (A) and peak II fractions
(B) separated by DEAE-TSK column chromatography.
Figure 3 presents DEAF-TSK column chromatography using a sodium
phosphate buffer of pH 6.5.
Figure 4 presents SDS-PAGE analysis of column fractions of peak I
(lane 1 ), peak II (lane 3) and peak III (lane 2) separated by DEAE-TSK column
chromatography at pH 6.5. Lane M denotes molecular weight standard markers.
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Figure 5 presents an HPLC chromatogram of a tryptic digest of peak II
protein from DEAE-TSK column chromatography at pH 6.5. The dotted line
indicates the concentration gradient of acetonitrile in the mobile phase.
Figure 6 presents a restriction enzyme map of the Lambda GEM 5- 1. The
1.53 kb DNA fragment (#SDH 2- 1 ) used as probe is shown as solid bar.
Figure 7 presents the locations of SI, S2 and S3 DNA fragments generated
with different sets of restriction enzymes from 5.7 kb Pstl fragment of Lambda
GEM 5-1.
Figure 8 presents the nucleotide sequence of 4,830 bases (SEQ ID N0:7)
of the 5.7 kb PstI fragment. The deduced amino acid sequence for the first and
the second subunit is shown below the nucleotide sequence. The N-terminal
amino acid sequence obtained by the N-terminal amino acid sequencing of the
purified sorbitol dehydrogenase is underlined. Signal sequence cleavage site
is
marked as a triangle. The heme-binding sequences are underlined with dotted
lines. Potential ribosome-binding sequences (SD) are enclosed in boxes. The
transcription termination stem-and-loop structure is indicated by arrows. The
complete coding sequence for the first subunit gene is located at position 665-
2,929 (SEQ ID NO:1 ), with the signal sequence located at position 665-766,
and
the coding sequence for the mature protein of the SDH first subunit located at
position 767-2,929 (SEQ ID N0:22). The complete coding sequence for the
second subunit gene is located at position 2,964-4,400 (SEQ ID N0:2), with the
signal sequence located at position 2,964-3,071, and the coding sequence for
the
mature protein of the SDH second subunit located at position 3,072-4,400 (SEQ
ID N0:23).
Figure 9 presents a restriction enzyme map of CIaI-#69. The closed box
represents the coding region of the third subunit gene of sorbitol
dehydrogenase.
Figure 10 presents the nucleotide sequence (SEQ ID N0:8) of DNA
fragment containing the third subunit gene of sorbitol dehydrogenase. The
deduced amino acid sequence is shown below the nucleotide sequence. Signal
sequence cleavage site is marked as a triangle. Potential ribosome-binding
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sequence (SD) is enclosed in box. The complete coding sequence for the third
subunit gene is located at position 1,384-2.304 (SEQ ID N0:3), with the signal
sequence located at position 1,384-1.461. and the coding sequence for the
mature
protein of the SDH third subunit located at position 1,462-2,304 (SEQ ID
N0:24).
Detailed Description of the Preferred Embodiments
1. Definitions
Cloning Vector: A plasmid or phage DNA or other DNA sequence
which is able to replicate autonomously in a host cell, and which is
characterized
by one or a small number of restriction endonuclease recognition sites at
which
such DNA sequences may be cut in a determinable fashion without loss of an
essential biological function of the vector. and into which a DNA fragment may
be spliced in order to bring about its replication and cloning. The cloning
vector
may further contain a marker suitable for use in the identification of cells
transformed with the cloning vector. Markers, for example, provide
tetracycline
resistance or ampicillin resistance.
Expression: Expression is the process by which a polypeptide is
produced from a structural gene. The process involves transcription of the
gene
into mRNA and the translation of such mRNA into polypeptide(s).
Expression Vector: A vector similar to a cloning vector but which is
capable of enhancing the expression of a gene which has been cloned into it,
after
transformation into a host. The cloned gene is usually placed under the
control
of (i.e., operably linked to) certain control sequences such as promoter
sequences.
Promoter sequences may be either constitutive or inducible.
Gene: A DNA sequence that contains information needed for expressing
a polypeptide or protein.
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Host: Any prokaryotic or eukaryotic cell that is the recipient of a
replicable expression vector or cloning vector. A "host," as the term is used
herein, also includes prokaryotic or eukaryotic cells that can be genetically
engineered by well known techniques to contain desired genes) on its
chromosome or genome. For examples of such hosts, see Sambrook et al.,
Molecular Cloning: A Laboratory _~lanual, Second Edition, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York ( 1989).
Homologous/Nonhomologous: Two nucleic acid molecules are
considered to be "homologous" if their nucleotide sequences share a similarity
of
greater than 50%, as determined b~~ HASH-coding algorithms (Wilber, W.J. and
Lipman, D.J., Proc. Natl. Acad. Sci. 80:726-730 (1983)). Two nucleic acid
molecules are considered to be "nonhomologous" if their nucleotide sequences
share a similarity of less than 50%.
Mutation: As used herein, the term refers to a single base pair change,
insertion or deletion in the nucleotide sequence of interest.
Mutagenesis: As used herein, the term refers to a process whereby a
mutation is generated in DNA. With "random" mutatgenesis, the exact site of
mutation is not predictable, occurring anywhere in the chromosome of the
microorganism, and the mutation is brought about as a result of physical
damage
caused by agents such as radiation or chemical treatment.
Operon: As used herein, the term refers to a unit of bacterial gene
expression and regulation, including the structural genes and regulatory
elements
in DNA.
Parental Strain: As used herein, the term refers to a strain of
microorganism subjected to some form of mutagenesis to yield the
microorganism of the invention.
Phenotype: As used herein, the term refers to observable physical
characteristics dependent upon the genetic constitution of a microorganism.
Promoter: A DNA sequence generally described as the 5' region of a
gene, located proximal to the start codon. The transcription of an adjacent
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genes) is initiated at the promoter region. If a promoter is an inducible
promoter,
then the rate of transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing agent if
the
promoter is a constitutive promoter.
Recombinant Host: According to the invention, a recombinant host may
be any prokaryotic or eukaryotic cell which contains the desired cloned genes
on
an expression vector or cloning vector. This term is also meant to include
those
prokaryotic or eukaryotic cells that have been genetically engineered to
contain
the desired genes) in the chromosome or genome of that organism.
Recombinant Vector: Any cloning vector or expression vector which
contains the desired cloned gene(s).
2. Isolation and Purification of Sorbitol DelZydrogenase
The present invention isolates and purifies SDH from the cytoplasmic
membrane of G. suboxydans KCTC (Korea Culture Type Collection) 2111
(equivalent to ATCC 621 ) using a series of column chromatographic steps.
Biochemical properties of the purified enzyme are provided, as well as the
isolation of each subunit and a determination of the N-terminal amino acid
sequence of each subunit using an amino acid sequence analyzer (Applied
Biosystems, 477A).
The newly characterized enzyme is different from the reported FAD-
dependent SDH from G. suboxydans IFO 3254 strain (Shinagawa, E. et al., Agric.
Biol. Chem., 46:135 (1982)), containing pyrroloquinoline quinone (PQQ) as a
cofactor and comprising three subunits (Choi, E. S. et al., FEMSMicrobiol.
Lett.,
125:45 (1995)).
The SDH of the invention may be isolated using standard protein
techniques. Briefly, G. suboxydans KCTC 2111 is cultured in SYP medium (5%
D-sorbitol, 1 % Bacto-Peptone, 0.5% yeast extract) and the cells are lysed in
a 10
mM sodium acetate buffer solution (pH 5.0). After centrifuged at 12,000 g to
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remove cell debris, the supernatant is centrifuged in an ultracentrifuge to
recover
cytoplasmic membrane fraction. Purification is completed by solubilizing the
cytoplasmic membrane fraction with 1.5% n-octylglucoside (Boehringer
Mannheim) and passage over a series of chromatographic columns, including
CM-TSK 650 (S) (Merck), DEAF-TSK 650 (S) (Merck), Mono-S (Pharmacia)
and Superose 12 (Pharmacia).
The purified enzyme is active to~i~ards polyols such as D-sorbitol ( 100%),
D-mannitol (68%) and D-ribitol (70%). Activity of the enzyme increases up to
nine fold when pyrroloquinoline quinone (PQQ) is added, suggesting that PQQ
is a cofactor for the enzyme; fluorescence spectrum analysis confirmed that
the
purified enzyme contains pyrroloquinoline quinone (PQQ). The absorption
spectrum analysis of the purified enzyme demonstrates that this enzyme
contains
cytochrome c. When the purified enzyme is subjected to polyacrylamide gel
electrophoresis (PAGE) at pH 4.3 (Reisfeld, R.A. et al., Nature,195:281 (
1962)),
it forms a single activity band after activity staining on the gel.
The initial SDS-PAGE analysis of the purified enzyme showed that the
enzyme comprised three subunits of 7~ kDa, 50 kDa and 14 kDa which were
named the first subunit, the second subunit, and the third subunit,
respectively
(Choi, E. S. et al., FEMSMicrobiol. Lett., 125:45 (1995)). In a further study
of
the enzyme, however, it was discovered that another subunit of 29 kDa played a
very important role in the stability and the catalytic activity of SDH. That
is,
while investigating a variety of pH conditions in an effort to increase
protein
separation capability during purification on DEAF-TSK column, it was
discovered that the partially resolved activity peaks eluting at pH 5.0 were
completely resolved into two separate activity peaks when eluted at pH 6Ø
The
early eluting activity peak quickly lost enzyme activity and the late eluting
activity peak remained stable. By SDS-PAGE analysis of these two peaks, it was
found that the peak with stable enzyme activity contained an additional
subunit
of 29 kDa in addition to the 75 and SO kDa subunits, whereas the one that
quickly
lost enzyme activity contained only 7~ and 50 kDa subunits. This additional
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subunit of 29 kDa was renamed the third subunit: it is uncertain whether the
14
kDa subunit previously assigned the third subunit is a true subunit of the
enzyme
when comparing the relative amount with other subunits on the acrylamide gel.
It was also found that a further increase of the pH of the elution buffer to
pH 6.5
resulted in a complete separation of three subunits into individual subunits.
When different combinations of two or three subunits were tested for the
restoration of enzyme activity by the Ferric-Dupanol assay method (Wood, W.A.
et al., Meth. Enzymol, 5:287 (1962)), it was found that enzyme activity was
fully
restored only in the presence of the third subunit of 29 kDa. Therefore, it
was
concluded that the third subunit of 29 kDa plays important roles in the
stability
and the catalytic activity of SDH.
The Michaelis-Menten constants. when using D-sorbitol as substrate, was
determined to be Km =~ 120 mM and Vmax = 3.9 x 10-5 M/min. Dichlorophenol
indophenol (DCIP) or ferricyanide worked effectively as electron acceptor of
the
enzyme. When phenazine methosulfate (PMS) was added as an electron
mediator, the enzyme activity increased. Calcium or magnesium ion addition
significantly increased purified enzyme activity, whereas copper ion addition
seriously inhibited activity.
Further details ofthe purification and characterization ofthe SDH enzyme
of the invention are provided in Example 1.
3. Nucleic Acid Molecules of the Invention
The invention provides isolated nucleic acid molecules encoding one or
more of the three subunits of the SDH enzyme described herein. Methods and
techniques designed for the manipulation of isolated nucleic acid molecules
are
well known in the art. For example, methods for the isolation, purification
and
cloning of nucleic acid molecules, as well as methods and techniques
describing
the use of eukaryotic and prokaryotic host cells and nucleic acid and protein
expression therein, are described by Sambrook, et al., Molecular Cloning: A
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Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y., 1989, and
Current Protocols in Molecular Biology. Frederick M. Ausubel et al. Eds., John
Wiley & Sons, Inc., 1987, the disclosure of which is hereby incorporated by
reference.
More particularly, the invention provides several isolated nucleic acid
molecules encoding the individual 75 kDa, 50 kDa, and 29 kDa subunit proteins
of SDH enzyme of the invention. Additionally, the invention provides several
isolated nucleic acid molecules encoding one or more of subunit proteins of
the
SDH enzyme of the invention. For the purposes of clarity, the particular
isolated
nucleic molecules of the invention are described. Thereafter, specific
properties
and characteristics of these isolated nucleic acid molecules are described in
more
detail.
Unless otherwise indicated, all nucleotide sequences determined by
sequencing a DNA molecule herein were determined using an automated DNA
sequencer (such as the Model 373A from Applied Biosystems, Inc.), and all
amino acid sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined by this
automated approach, any nucleotide sequence determined herein may contain
some errors. Nucleotide sequences determined by automation are typically at
least about 90% identical, more typically at least about 95% to at least about
99.9% identical to the actual nucleotide sequence of the sequenced DNA
molecule. The actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in the art.
As is also known in the art, a single insertion or deletion in a determined
nucleotide sequence compared to the actual sequence will cause a frame shift
in
translation of the nucleotide sequence such that the predicted amino acid
sequence encoded by a determined nucleotide sequence will be completely
different from the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or deletion.
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In one embodiment, the invention provides an isolated nucleic acid
molecule for the first (75 kDa) subunit of the SDH enzyme of the invention
comprising a polynucleotide sequence selected from the group consisting of (a)
the polynucleotide of SEQ ID NO: l ; (b) a polynucleotide fragment at least
about
20 nucleotides in length of the polvnucleotide of SEQ ID NO:1; (c) a
polynucleotide encoding the amino acid sequence of SEQ ID N0:4; and (d) a
polynucleotide encoding a fragment at least about 10 amino acids in length of
the
amino acid sequence of SEQ ID N0:4.
In another embodiment, the invention provides an isolated nucleic acid
molecule for the first (75 kDa) subunit of the SDH enzyme of the invention
comprising a polynucleotide at least about 95% identical to the isolated
nucleic
acid sequence for the first (75 kDa) subunit of the SDH enzyme of the
invention
described above.
Another embodiment of the invention provides an isolated nucleic acid
molecule for the second (50 kDa) subunit of the SDH enzyme of the invention
comprising a polynucleotide sequence selected from the group consisting of:
(a)
the polynucleotide, or fragment thereof, of SEQ ID N0:2; (b) a polynucleotide
fragment at least about 20 nucleotides in length of the polynucleotide of SEQ
ID
N0:2; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO:S;
and (d) a polynucleotide encoding a fragment at least about 10 amino acids in
length of the amino acid sequence of SEQ ID NO:S.
In another embodiment, the invention provides an isolated nucleic acid
molecule for the second (50 kDa) subunit of the SDH enzyme of the invention
comprising a polynucleotide at least about 95% identical to the isolated
nucleic
acid sequence for the second (50 kDa) subunit of the SDH enzyme of the
invention described above.
Another embodiment of the invention provides an isolated nucleic acid
molecule for the third (29 kDa) subunit of the SDH enzyme of the invention
comprising a polynucleotide sequence selected from the group consisting of:
(a)
the polynucleotide of SEQ ID N0:3; (b)a polynucleotide fragment at least about
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20 nucleotides in length of the polynucleotide of SEQ ID N0:3; (c) a
polynucleotide encoding the amino acid sequence of SEQ ID N0:6; and (d) a
polynucleotide encoding a fragment at least about 10 amino acids in length of
the
amino acid sequence of SEQ ID N0:6.
In another embodiment, the invention provides an isolated nucleic acid
molecule for the third (29 kDa) subunit of the SDH enzyme of the invention
comprising a polynucleotide at least about 95% identical to the isolated
nucleic
acid sequence for the third (29 kDa) subunit of the SDH enzyme of the
invention
described above.
Another embodiment the invention provides an isolated nucleic acid
molecule encoding both the first (75 kDa) and second (50 kDa) subunit proteins
of the SDH enzyme of the invention comprising a polynucleotide sequence
selected from the group consisting of: (a) the polynucleotide of SEQ ID N0:7;
and (b) a polynucleotide fragment at least about 20 nucleotides in length of
the
polynucleotide of SEQ ID N0:7.
In another embodiment, the invention provides an isolated nucleic acid
molecule for the first (75 kDa) and second (50 kDa) subunit proteins of the
SDH
enzyme of the invention comprising a polynucleotide at least about 95%
identical
to the isolated nucleic acid sequence for the first (75 kDa) and second (50
kDa)
subunit proteins of the SDH enzyme of the invention.
Another embodiment of the invention provides an isolated nucleic acid
molecule for the third (29 kDa) subunit of the SDH enzyme of the invention
comprising a polynucleotide sequence selected from the group consisting o~ (a)
the polynucleotide of SEQ ID N0:8; and (b) a polynucleotide fragment at least
about 20 nucleotides in length of the polynucleotide of SEQ ID N0:8.
In another embodiment, the invention provides an isolated nucleic acid
molecule for the third (29 kDa) subunit of the SDH enzyme of the invention
comprising an isolated nucleic acid molecule at least about 95% identical to
the
isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH
enzyme
of the invention described above.
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By "isolated" nucleic acid molecule is intended a nucleic acid molecule,
DNA or RNA, which has been removed from its native environment. For
example, recombinant DNA molecules contained in a vector are considered
isolated for the purposes of the present invention. Further examples of
isolated
DNA molecules include recombinant DNA molecules maintained in heterologous
host cells or purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA
molecules of the present invention. Isolated nucleic acid molecules according
to
the present invention further include such molecules produced synthetically.
RNA vectors may also be utilized with the SDH nucleic acid molecules
disclosed in the invention. These vectors are based on positive or negative
strand
RNA viruses that naturally replicate in a wide variety of eukaryotic cells
(Bredenbeek, P.J. and Rice, C.M., I irology 3:297-310 (1992)). Unlike
retroviruses, these viruses lack an intermediate DNA life-cycle phase,
existing
entirely in RNA form. For example, alpha viruses are used as expression
vectors
for foreign proteins because they can be utilized in a broad range of host
cells and
provide a high level of expression; examples of viruses of this type include
the
Sindbis virus and Semliki Forest virus (Schlesinger, S., TIBTECH 11:18-22
(1993); Frolov, L, et al., Proc. Natl. Acad. Sci. (USA) 93:11371-11377
(1996)).
As exemplified by Invitrogen's Sinbis expression system, the investigator may
conveniently maintain the recombinant molecule in DNA form (pSinrep5
plasmid) in the laboratory, but propagation in RNA form is feasible as well.
In
the host cell used for expression, the vector containing the gene of interest
exists
completely in RNA form and may be continuously propagated in that state if
desired.
In another embodiment, the invention further provides variant nucleic acid
molecules that encode portions, analogs or derivatives of the isolated nucleic
acid
molecules described herein. Variants include those produced by nucleotide
substitutions, deletions or additions, which may involve one or more
nucleotides.
The variants may be altered in coding regions, non-coding regions, or both.
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Alterations in the coding regions may produce conservative or non-conservative
amino acid substitutions, deletions or additions.
Variants of the isolated nucleic acid molecules ofthe invention may occur
naturally, such as a natural allelic variant. By an "allelic variant" is
intended one
of several alternate forms of a gene occupying a given locus on a chromosome
of
an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known mutagenesis
techniques.
Isolated nucleic acid molecules of the invention also include
polynucleotide sequences that are 95%. 96%, 97%, 98% and 99% identical to the
isolated nucleic acid molecules described herein. Computer programs such as
the
BestFit program (Wisconsin Sequence Analysis Package, Version 10 for Unix,
Genetics Computer Group, Universiy Research Park, 575 Science Drive,
Madison, WI 53711) may be used to determine whether any particular nucleic
acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to the
nucleotide
sequences disclosed herein or the the nucleotides sequences of the deposited
clones described herein. BestFit uses the local homology algorithm of Smith
and
Waterman, Advances in Applied Mathematics 2: 482-489 ( 1981 ), to find the
best
segment of homology between two sequences.
By way of example, when a computer alignment program such as BestFit
is utilized to determine 95% identity to a reference nucleotide sequence, the
percentage of identity is calculated over the full length of the reference
nucleotide
sequence and gaps in homology of up to 5% of the total number of nucleotides
in the reference sequence are allowed. Thus, per 100 base pairs analyzed, 95%
identity indicates that as many as 5 of 100 nucleotides in the subject
sequence
may vary from the reference nucleotide sequence.
The invention also encompasses fragments of the nucleotide sequences
and isolated nucleic acid molecules described herein. In a preferred
embodiment
the invention provides for fragments that are at least 20 bases in length. The
length of such fragments may be easily defined algebraically. For example, SEQ
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ID NO:1 provides for an isolated nucleotide molecule that is 2, 265 bases in
length. A fragment (F 1 ) of SEQ ID NO:1 at least 20 bases in length may be
defined as F 1= 20 + X, wherein X is defined to be zero or any whole integer
from
1 to 2,245. Similarly, fragments for other isolated nucleic acid molecules
described herein may be defined as follows: for SEQ ID N0:2 which is 1,437
bases in length, a fragment (F2) of SEQ ID N0:2 at least 20 bases in length
may
be defined as F2= 20 + X, wherein X is defined to be zero or any whole integer
from 1 to 1,417; for SEQ ID N0:3 which is 921 bases in length, a fragment (F3)
of SEQ ID N0:3 at least 20 bases in length may be defined as F3= 20 + X,
wherein X is defined to be zero or anv whole integer from 1 to 901; for SEQ ID
N0:7 which is 4,830 bases in length, a fragment (F7) of SEQ ID N0:7 at least
20
bases in length may be defined as F7= 20 + X, wherein X is defined to be zero
or
any whole integer from 1 to 4,810; and for SEQ ID N0:8 which is 2,700 bases in
length, a fragment (F8) of SEQ ID N0:8 at least 20 bases in length may be
defined as F8= 20 + X, wherein X is defined to be zero or any whole integer
from
1 to 2,680. As will be understood by those skilled in the art, the isolated
nucleic
acid sequence fragments of the invention may single stranded or double
stranded
molecules.
The invention discloses isolated nucleic acid sequences encoding the three
subunit proteins of the SDH enzyme of the invention. Computer analysis
provides information regarding the open reading frames, putative signal
sequence
and mature protein forms of each subunit. Genes encoding the first (75 kDa)
and
second (50 kDa) subunits are completely contained in a 5.7 kb Pst I fragment
of
the lambda GEM 5-1 clone, which is deposited in bacteria under the accession
number KCTC 0593BP and as DNA under accession number KCTC 0597BP
with the Korean Collection for Type Cultures (KCTC), Korea Research Institute
of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong, Yusong-Ku, Taejon
305-33, Republic of Korea. The third (29 kDa) subunit gene is contained in a
sequence 4.5 kb in length, referred to as Cla I-#69, which is deposited in
bacteria
under the accession number KCTC 0594BP and as DNA under accession number
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KCTC 0598BP with the Korean Collection for Type Cultures (KCTC), Korea
Research Institute of Bioscience and Biotechnology (KRIBB), 52, Oun-Dong,
Yusong-Ku, Taejon 305-33, Republic of Korea.
Thus, the invention provides an isolated nucleic acid molecule (KCTC
0597BP) carried in the novel strain KCTC 0593BP, and the invention also
provides an isolated nucleic acid molecule (KCTC 0598BP) carried in the novel
strain KCTC 0594BP.
Sequence obtained from the lambda GEM 5-1, 5.7 kb Pst I fragment is
presented in Figure 8 and assigned SEQ ID N0:7. The complete coding sequence
for the first subunit gene is located at position 665-2,929 (SEQ ID NO:1),
with
the signal sequence located at position 665-766, and the coding sequence for
the
mature protein of the SDH first subunit located at position 767-2,929 (SEQ ID
N0:22). The complete coding sequence for the second subunit gene is located
at position 2,964-4,400 (SEQ ID N0:2), with the signal sequence located at
position 2,964-3,071, and the coding sequence for the mature protein of the
SDH
second subunit located at position 3,072-4,400 (SEQ ID N0:23).
Thus, another embodiment of the invention provides isolated nucleic acid
molecules derived from sequence obtained from the lambda GEM 5-1, 5.7 kb Pst
I fragment that is presented in Figure 8 and identified as SEQ ID N0:7. Such
isolated nucleic acid molecules include the following: (1) nucleotides 1-664
of
SEQ ID N0:7 identified as SEQ ID I~T0:28; (2) nucleotides 50-664 of SEQ ID
N0:7 identified as SEQ ID N0:29; (3) nucleotides 100-664 of SEQ ID N0:7
identified as SEQ ID N0:30; (4) nucleotides 150-664 of SEQ ID N0:7 identified
as SEQ ID N0:31; (5) nucleotides 200-664 of SEQ ID N0:7 identified as SEQ
ID N0:32; (6) nucleotides 250-664 of SEQ ID N0:7 identified as SEQ ID
N0:33; (7) nucleotides 300-664 of SEQ ID N0:7 identified as SEQ ID N0:34;
(8) nucleotides 350-664 of SEQ ID N0:7 identified as SEQ ID N0:35; (9)
nucleotides 400-664 of SEQ ID N0:7 identified as SEQ ID N0:36; (10)
nucleotides 450-664 of SEQ ID N0:7 identified as SEQ ID N0:37; (11)
nucleotides 500-664 of SEQ ID N0:7 identified as SEQ ID N0:38; (12)
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nucleotides 550-664 of SEQ ID N0:7 identified as SEQ ID N0:39; (13)
nucleotides 600-664 of SEQ ID N0:7 identified as SEQ ID N0:40; (14) the
nucleotide sequence encoding the full length subunit 1 protein of the SDH of
the
invention from nucleotides 665-2,929 of SEQ ID N0:7 identified as SEQ ID
NO:1; (15) the nucleotide sequence encoding the mature form of the subunit 1
protein of the SDH of the invention from nucleotides 767-2,929 of SEQ ID N0:7
identified as SEQ ID N0:22; (16) the nucleotide sequence encoding the full
length subunit 2 protein of the SDH of the invention from nucleotides 2,964-
4,400 of SEQ ID N0:7 identified as SEQ ID N0:2; (17) the nucleotide sequence
encoding the mature form of the subunit 2 protein of the SDH of the invention
from nucleotides3,072-4,400 of SEQ ID N0:7 identified as SEQ ID N0:23; (18)
nucleotides 2,930-2,963 of SEQ ID NT0:7 identified as SEQ ID N0:41; (19)
nucleotides 4,401-4,451 of SEQ ID N0:7 identified as SEQ ID N0:42; (20)
nucleotides 4,401-4,501 of SEQ ID I~T0:7 identified as SEQ ID N0:43; (21)
nucleotides 4,401-4,551 of SEQ ID Iv'0:7 identified as SEQ ID N0:44; (22)
nucleotides 4,401-4,601 of SEQ ID IvT0:7 identified as SEQ ID N0:45; (23)
nucleotides 4,401-4,651 of SEQ ID N0:7 identified as SEQ ID N0:46; (24)
nucleotides 4,401-4,701 of SEQ ID N0:7 identified as SEQ ID N0:47; (25)
nucleotides 4,401-4,751 of SEQ ID N0:7 identified as SEQ ID N0:48; (26)
nucleotides 4,401-4,801 of SEQ ID N0:7 identified as SEQ ID N0:49; and (27)
nucleotides 4,401-4,830 of SEQ ID N0:7 identified as SEQ ID NO:50.
The sequence obtained from the Cla I-#69 clone is presented in Figure 10
and assigned SEQ ID N0:8. The complete coding sequence for the third subunit
gene is located at position 1,384-2,304 (SEQ ID N0:3), with the signal
sequence
located at position 1,384-1,461, and the coding sequence for the mature
protein
of the SDH third subunit located at position 1,462-2,304 (SEQ ID N0:24).
Thus, another embodiment of the invention provides isolated nucleic acid
molecules derived from sequence obtained from the Cla I-#69 clone that is
presented in Figure 10 and assigned SEQ ID N0:8. Such isolated nucleic acid
molecules include the following: (1) nucleotides 1-1,383 of SEQ ID N0:8
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identified as SEQ ID N0:51; (2) nucleotides 50-1,383 of SEQ ID N0:8 identified
as SEQ ID N0:52; (3) nucleotides 100-1,383 of SEQ ID N0:8 identified as SEQ
ID N0:53; (4) nucleotides 150-1,383 of SEQ ID N0:8 identified as SEQ ID
N0:54; (5) nucleotides 200-1,383 of SEQ ID N0:8 identified as SEQ ID N0:55;
(6) nucleotides 250-1,383 of SEQ ID N0:8 identified as SEQ ID N0:56; (7)
nucleotides 300-1,383 of SEQ ID identifiedSEQ ID N0:57;(8)
N0:8 as
nucleotides 350-1,383 of SEQ ID identifiedSEQ ID N0:58;(9)
N0:8 as
nucleotides 400-1,383 of SEQ ID identifiedSEQ ID N0:59;(10)
N0:8 as
nucleotides 450-1,383 of SEQ ID identifiedSEQ ID N0:60;(11)
N0:8 as
nucleotides 500-1,383of SEQ ID identifiedSEQ ID N0:61;(12)
N0:8 as
nucleotides 550-1,383 of SEQ ID identifiedSEQ ID N0:62;(13)
N0:8 as
nucleotides 600-1,383 of SEQ ID identifiedSEQ ID N0:63;(14)
N0:8 as
nucleotides 600-1,383 of SEQ ID identifiedSEQ ID N0:64;(15)
N0:8 as
nucleotides 650-1,383 of SEQ ID identifiedSEQ ID N0:65;(16)
N0:8 as
nucleotides 700-1,383of SEQ ID identifiedSEQ ID N0:66;(17)
N0:8 as
nucleotides 750-1,383 of SEQ ID identifiedSEQ ID N0:67;(18)
N0:8 as
nucleotides 800-1,383 of SEQ ID identifiedSEQ ID N0:68;(19)
N0:8 as
nucleotides 850-1,383 of SEQ ID identifiedSEQ ID N0:69;(20)
N0:8 as
nucleotides 900-1,383 of SEQ ID identifiedSEQ ID N0:70;(21)
N0:8 as
nucleotides 950-1,383of SEQ ID identifiedSEQ ID N0:71;(22)
N0:8 as
nucleotides 1,000-1,383 of SEQ ID N0:8 identified as SEQ
ID N0:72; (23)
nucleotides 1,050-1,383 of SEQ ID N0:8 identified as SEQ
ID N0:73; (24)
nucleotides 1,100-1,383 of SEQ ID N0:8 identified as SEQ
ID N0:74; (25)
nucleotides 1,150-1,383 of SEQ ID N0:8 identified as SEQ
ID N0:75; (26)
nucleotides 1,200-1,383 of SEQ ID N0:8 identified as SEQ
ID N0:76; (27)
nucleotides 1,250-1,383 of SEQ ID N0:8 identified as SEQ ID N0:77; (28)
nucleotides 1,300-1,383 of SEQ ID N0:8 identified as SEQ ID N0:78; (29)
nucleotides 1,350-1,383 of SEQ ID N0:8 identified as SEQ ID N0:79;(30) the
nucleotide sequence encoding the full length SDH subunit 3 protein of the
invention from nucleotides 1,384-1,461 of SEQ ID N0:8 identified as SEQ ID
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N0:3; (31 ) the nucleotide sequence encoding the mature form of the SDH
subunit
3 protein of the invention from nucleotides 1,462-2,304 of SEQ ID N0:8
identified as SEQ ID N0:24; (32) nucleotides 2,305-2,355 of SEQ ID N0:8
identified as SEQ ID N0:80; (33) nucleotides 2,305-2,405 of SEQ ID N0:8
identified as SEQ ID N0:81; (34) nucleotides 2,305-2,455 of SEQ ID N0:8
identified as SEQ ID N0:82; (35) nucleotides 2,305-2,505 of SEQ ID N0:8
identified as SEQ ID N0:83; (32) nucleotides 2,305-2,555 of SEQ ID N0:8
identified as SEQ ID N0:84; (32) nucleotides 2,305-2,605 of SEQ ID N0:8
identified as SEQ ID N0:85; (32) nucleotides 2,305-2,655 of SEQ ID N0:8
identified as SEQ ID N0:86; and (33) nucleotides 2,305-2,700 of SEQ ID N0:8
identified as SEQ ID N0:87.
The invention also includes recombinant constructs comprising one or
more of the sequences as broadly described above. The constructs comprise a
vector, such as a plasmid or viral vector, into which a sequence of the
invention
has been inserted, in a forward or reverse orientation. In a preferred aspect
of this
embodiment, the construct further comprises regulatory sequences, including,
for
example, a promoter, operably linked to the sequence. Large numbers of
suitable
vectors and promoters are known to those of skill in the art and are
commercially
available. The following vectors are provided by way of example: Bacterial-
pET (Novagen), pQE70, pQE60, pQE-9 (Qiagen), pBs, phagescript, psiXl74,
pBlueScript SK, pBsKS, pNHBa, pNHl6a, pNHl8a, pNH46a (Stratagene);
pTrc99A, pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia); and Eukaryotic
pWLneo, pSV2cat, pOG44, pXTI, pSG (Stratagene) pSVK3, pBPV, pMSG,
pSVL (Pharmacia). Thus, these and any other plasmids or vectors may be used
as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable
markers.
Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial
promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic
promoters include CMV immediate early, HSV thymidine kinase, early and late
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SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of ordinary skill in
the
art.
In another embodiment, the invention provides processes for producing
the vectors described herein which comprises: (a) inserting the isolated
nucleic
acid molecule of the invention into a vector; and (b) selecting and
propagating
said vector in a host cell.
Representative examples of appropriate hosts include, but are not limited
to, bacterial cells, such as Gluconobacter, Brevibacterium, Corynebacterim, E.
coli, Streptomyces, Salmonella typhimurium, Acetobacter, Pseudomonas,
Pseudogluconobacter, Bacillus and Agrobacterium cells; fungal and yeast
organisms including Saccharomyces, Kl uyveromyces, Aspergillus and Rhizopus;
insect cells such as Drosophila S2 and Spodoptera Sf~ cells; animal cells such
as
CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture
mediums and conditions for the above-described host cells are known in the
art.
4. Polypeptides of the Invention
The invention provides isolated polypeptide molecules for the SDH
enzyme of the invention. Methods and techniques designed for the manipulation
of isolated polypeptide molecules are well known in the art. For example,
methods for the isolation and purification of polypeptide molecules are
described
Current Protocols in Protein Science, John E. Coligan et al. Eds., John Wiley
&
Sons, Inc., 1997, the disclosure of which is hereby incorporated by reference.
More particularly, the invention provides several isolated polypeptide
molecules encoding the individual 75 kDa, 50 kDa, and 29 kDa subunit proteins
of SDH enzyme of the invention. For the purposes of clarity, the particular
isolated polypeptide molecules of the invention are described. Thereafter,
specific properties and characteristics of these isolated polypeptide
molecules are
described in more detail.
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In one embodiment, the invention provides an isolated polypeptide
comprising a polypeptide sequence selected from the group consisting of: (a)
the
polypeptide sequence encoded in the polynucleotide sequence of SEQ ID NO:1;
(b) the polypeptide sequence of SEQ ID N0:4; and (c) a polypeptide at least
about 10 amino acids long from the polypeptide sequence of SEQ ID N0:4.
In another embodiment, the invention provides an isolated polypeptide
comprising a polypeptide sequence selected from the group consisting of: (a)
the
polypeptide sequence encoded in the polynucleotide sequence of SEQ ID N0:2;
(b) the polypeptide sequence of SEQ ID NO:S; and (c) a polypeptide at least
about 10 amino acids long from the polypeptide sequence of SEQ ID NO:S.
In yet another embodiment, the invention provides an isolated polypeptide
comprising a polypeptide sequence selected from the group consisting of: (a)
the
polypeptide sequence encoded in the polynucleotide sequence of SEQ ID N0:3;
(b) the polypeptide sequence of SEQ ID N0:6; and (c) a polypeptide at least
about 10 amino acids long from the polypeptide sequence of SEQ ID N0:6.
Other embodiments of the invention include an isolated polypeptide
sequence comprising the polypeptide encoded by the isolated nucleic acid
sequence SEQ ID N0:7; an isolated polypeptide sequence comprising the
polypeptide encoded by the isolated nucleic acid sequence SEQ ID N0:8; an
isolated polypeptide sequence comprising the polypeptide encoded by the DNA
clone contained in KCTC Deposit No. 0593BP; and an isolated polypeptide
sequence comprising the polypeptide encoded by the DNA clone contained in
KCTC Deposit No. 0594BP.
The term "isolated polypeptide" is used herein to mean a polypeptide
removed from its native environment. Thus, a polypeptide produced and/or
contained within a recombinant host cell is considered isolated for purposes
of the
present invention. Also intended as an "isolated polypeptide" are polypeptides
that have been purified, partially or substantially, from a recombinant host
cell.
Polypeptides of the present invention include naturally purified products,
products of chemical synthetic procedures, and products produced by
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recombinant techniques from a prokaryotic or eukaryotic host, including, for
example, bacterial, yeast, higher plant. insect and mammalian cells. Depending
upon the host employed in a recombinant production procedure, the polypeptides
of the present invention may be glycosvlated or may be non-glycosylated. In
S addition, polypeptides of the invention may also include an initial modified
methionine residue, in some cases as a result of host-mediated processes.
The isolated polypeptides of the invention also include variants of those
polypeptides described above. The term "variants" is meant to include natural
allelic variant polypeptide sequences possessing conservative or
nonconservative
amino acid substitutions, deletions or insertions. The term "variants" is also
meant to include those isolated polypeptide sequences produced by the hand of
man, through known mutagenesis techniques or through chemical synthesis
methodology. Such man-made variants may include polypeptide sequences
possessing conservative or non-conservative amino acid substitutions,
deletions
or insertions.
Whether a particular amino acid is conservative or non-conservative is
well known to those skilled in the art. Conservative amino acid substitutions
do
not significantly affect the folding or activity of the protein. For exemplary
purposes, Table 1 presents a list of conservative amino acid substitutions.
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TABLE 1. Conservative Amino Acid Substitutions
Aromatic Phenylalanine
Tryptophan
Tyrosine
HydrophobicLeucine
Isoleucine
Valine
Polar Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic Aspartic
Acid
Glutamic
Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
Amino acids in the protein of the present invention that are essential for
function can be identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)).
Isolated polypeptide molecules of the invention also include polypeptide
sequences that are 95%, 96%, 97%. 98% and 99% identical to the isolated
polypeptide molecules described herein. Computer programs such as the BestFit
program (Wisconsin Sequence Analysis Package, Version 10 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive, Madison, WI
53711 ) may be used to determine whether any particular polypeptide molecule
is
at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequences
disclosed herein or the the polypeptide sequences encoded by the isolated DNA
molecule of the deposited clones described herein. BestFit uses the local
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homology algorithm of Smith and Waterman, Advances in Applied Mathematics
2: 482-489 ( 1981 ), to find the best segment of homology between two
sequences.
By way of example, when a computer alignment program such as BestFit
is utilized to determine 95% identity to a reference polypeptide sequence, the
percentage of identity is calculated over the full length of the reference
polypeptide sequence and gaps in homology of up to 5% of the total number of
amino acids in the reference sequence are allowed. Thus, per 100 amino acids
analyzed, 95% identity indicates that as many as 5 of 100 amino acids in the
subject sequence may vary from the reference polypeptide sequence.
The invention also encompasses fragments of the polypeptide sequences
and isolated polypeptide molecules described herein. In a preferred embodiment
the invention provides for fragments that are at least 10 amino acids in
length.
The length of such fragments may be easily defined algebraically. For example,
SEQ ID N0:4 provides for an isolated polypeptide molecule that is 754 amino
acids in length. A fragment (F4) of SEQ ID N0:4 at least 10 amino acids in
length may be defined as F4= 10 + X, wherein X is defined to be zero or any
whole integer from 1 to 744. Similarly. fragments for other isolated
polypeptide
molecules described herein may be defined as follows: for SEQ ID NO:S which
is 478 amino acids in length, a fragment (FS) of SEQ ID NO:S at least 10 amino
acids in length may be defined as FS= 10 + X, wherein X is defined to be zero
or
any whole integer from 1 to 468; and for SEQ ID N0:6 which is 306 amino acids
in length, a fragment (F6) of SEQ ID N0:6 at least 10 amino acids in length
may
be defined as F6= 10 + X, wherein X is defined to be zero or any whole integer
from 1 to 296.
Particularly preferred embodiments of the invention provide isolated
polypeptides such as the following: ( 1 ) the full length polypeptide the SDH
subunit 1 of the invention, encoded by the isolated nucleic acid molecule of
SEQ
ID NO: l and identified by SEQ ID N0:4; (2) the full length polypeptide the
SDH
subunit 2 of the invention, encoded by the isolated nucleic acid molecule of
SEQ
ID N0:2 and identified by SEQ ID NO:S; (3) the full length polypeptide the SDH
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subunit 3 of the invention, encoded by the isolated nucleic acid molecule of
SEQ
ID N0:3 and identified by SEQ ID N0:6; (4) the mature form of the SDH subunit
1 polypeptide of the invention, encoded by the isolated nucleic acid molecule
of
SEQ ID N0:22 and identified by SEQ ID N0:25; (5) the mature form of the SDH
subunit 2 polypeptide of the invention. encoded by the isolated nucleic acid
molecule of SEQ ID N0:24 and identified as SEQ ID N0:26; and the mature
form of the SDH subunit 3 polypeptide of the invention, encoded by the
isolated
nucleic acid molecule of SEQ ID N0:23 and identified as SEQ ID N0:27.
The invention also provides a process for producing a polypeptide
comprising: (a) growing the host cell containing the isolated nucleic acid
molecule of SEQ ID NO:1 or variants thereof; (b) expressing the polypeptide
encoded by said isolated nucleic acid molecule; and (c) isolating said
polypeptide.
In another embodiment, the invention provides a process for producing a
polypeptide comprising: (a) growing the host cell containing the isolated
nucleic
acid molecule of SEQ ID N0:2 or variants thereof; (b) expressing the
polypeptide
encoded by said isolated nucleic acid molecule; and (c) isolating said
polypeptide.
Another process provided by the invention is for the production of a
polypeptide which comprises: (a) growing the host cell containing the isolated
nucleic acid molecule of SEQ ID N0:3 or variants thereof; (b) expressing the
polypeptide encoded by said isolated nucleic acid molecule; and (c) isolating
said
polypeptide.
Representative examples of appropriate hosts include, but are not limited
to, bacterial cells, such as Gluconobacter, Brevibacterium, Corynebacterim, E.
coli, Streptomyces, Salmonella typhimurium, Acetobacter, Pseudomonas,
Pseudogluconobacter, Bacillus and Agrobacterium cells; fungal and yeast
organisms including Saccharomyces, Kluyveromyces, Aspergillus and Rhizopus;
insect cells such as Drosophila S2 and Spodoptera Sf~ cells; animal cells such
as
CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture
mediums and conditions for the above-described host cells are known in the
art.
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The polypeptide may be expressed in a modified form, such as a fusion
protein, and may include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of additional amino
acids,
particularly charged amino acids. may be added to the N-terminus of the
polypeptide to improve stability and persistence in the host cell, during
purification, or during subsequent handling and storage. Also, peptide
moieties
may be added to the polypeptide to facilitate purification. Such regions may
be
removed prior to final preparation of the polypeptide. The addition of peptide
moieties to polypeptides to engender secretion or excretion, to improve
stability
and to facilitate purification, among others, are familiar and routine
techniques
in the art.
Polypeptides of the invention may be recovered and purified from
recombinant cell cultures by well-know methods including ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography and
lectin chromatography. Most preferably, high performance liquid
chromatography ("HPLC") is employed for purification.
S. Production of L-Sorbose and 2-Keto-L-Gulonic Acid
The invention provides processes for the production of L-sorbose and
2-keto-L-gulonic acid, which are useful in the production of vitamin C.
In one embodiment, the invention provides a process for the production
of L-sorbose from D-sorbitol comprising: (a) transforming a host cell with at
least
one isolated nucleotide sequence selected from the group consisting o~ (i) a
polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1; (ii) a
polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2; and
(iii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3;
and (b) selecting and propagating said transformed host cell.
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In another embodiment, the invention provides a process for the
production of 2-keto-L-gulonic acid comprising: (a) transforming a host cell
with
at least one isolated nucleotide sequence selected from the group consisting
of:
(i) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1;
(ii)
a polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2; and
(iii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3;
and (b) selecting and propagating said transformed host cell.
Suitable bacteria for use as host cells in the processes provided herein for
the production of L-sorbose and 2-keto-L-gulonic acid are known to those
skilled
in the art. Such bacteria include, but are not limited to, Escherichia coli,
Brevibacterium, Corynebacterium, Gluconobacter, Acetobacter, Pseudomonas
and Pseudogluconobacter.
Other host cells for expression of the SDH enzyme of the invention
include: strains identified in U.S. Patent NO. 5,834,231; Glucanobacter
oxydans
DSM 4025 (U.S. Patent No. 4,960,690; Gluconobactor oxydans TI00 (Appl.
Environ. Microbiol. 63:454-460 (1997)); Pseudogluconobacter
saccharoketogenes IFO 14464 (European Patent No. 221 707); Pseudomonas
sorbosoxidans (U.S. Patent No. 4,933,289); and Acetobacter liquefaciens IFO
12258 (Appl Environ. Microbiol. 61:413-420 (1995)).
In other embodiments of the invention, a variety of fermentation
techniques known in the art may be employed in processes of the invention
drawn
to the production of L-sorbose and 2-keto-L-gulonic acid. Generally, L-sorbose
and 2-keto-L-gulonic acid may be produced by fermentation processes such as
the
batch type or of the fed-batch type. In batch type fermentations, all
nutrients are
added at the beginning of the fermentation. In fed-batch or extended fed-batch
type fermentations one or a number of nutrients are continuously supplied to
the
culture, right from the beginning of the fermentation or after the culture has
reached a certain age, or when the nutrients) which are fed were exhausted
from
the culture fluid. A variant of the extended batch of fed-batch type
fermentation
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is the repeated fed-batch or fill-and-draw fermentation, where part ofthe
contents
of the fermenter is removed at some time. for instance when the fermenter is
full,
while feeding of a nutrient is continued. In this way a fermentation can be
extended for a longer time.
Another type of fermentation, the continuous fermentation or chemostat
culture, uses continuous feeding of a complete medium, while culture fluid is
continuously or semi-continuously withdrawn in such a way that the volume of
the broth in the fermenter remains approximately constant. A continuous
fermentation can in principle be maintained for an infinite time.
In a batch fermentation an organism grows until one of the essential
nutrients in the medium becomes exhausted, or until fermentation conditions
become unfavorable (e.g. the pH decreases to a value inhibitory for microbial
growth). In fed-batch fermentations measures are normally taken to maintain
favorable growth conditions, e.g. by using pH control, and exhaustion of one
or
more essential nutrients is prevented by feeding these nutrients) to the
culture.
The microorganism will continue to grow, at a growth rate dictated by the rate
of
nutrient feed. Generally a single nutrient, very often the carbon source, will
become limiting for growth. The same principle applies for a continuous
fermentation, usually one nutrient in the medium feed is limiting, all other
nutrients are in excess. The limiting nutrient will be present in the culture
fluid
at a very low concentration, often unmeasurably low. Different types of
nutrient
limitation can be employed. Carbon source limitation is most often used. Other
examples are limitation by the nitrogen source, limitation by oxygen,
limitation
by a specific nutrient such as a vitamin or an amino acid (in case the
microorganism is auxotrophic for such a compound), limitation by sulphur and
limitation by phosphorous.
Illustrative examples of suitable supplemental carbon sources include, but
are not limited to: other carbohydrates, such as glucose, fructose, mannitol,
starch
or starch hydrolysate, cellulose hydrolysate and molasses; organic acids, such
as
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acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric
acid, and
fumaric acid; and alcohols, such as glycerol.
Illustrative examples of suitable nitrogen sources include, but are not
limited to: ammonia, including ammonia gas and aqueous ammonia; ammonium
salts of inorganic or organic acids, such as ammonium chloride, ammonium
nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea;
nitrate or nitrite salts, and other nitrogen-containing materials, including
amino
acids as either pure or crude preparations, meat extract, peptone, fish meal,
fish
hydrolysate, corn steep liquor, casein hydrolysate, soybean cake hydrolysate,
yeast
extract, dried yeast, ethanol-yeast distillate, soybean flour, cottonseed
meal, and
the like.
Illustrative examples of suitable inorganic salts include, but are not
limited to: salts of potassium, calcium, sodium, magnesium, manganese, iron,
cobalt, zinc, copper and other trace elements, and phosphoric acid.
Illustrative examples of appropriate trace nutrients, growth factors, and the
like include, but are not limited to: coenzyme A, pantothenic acid, biotin,
thiamine, riboflavin, flavine mononucleotide, flavine adenine dinucleotide,
other
vitamins, amino acids such as cysteine, sodium thiosulfate, p-aminobenzoic
acid,
niacinamide, and the like, either as pure or partially purified chemical
compounds
or as present in natural materials. Cultivation of the inventive microorganism
strain may be accomplished using any of the submerged fermentation techniques
known to those skilled in the art, such as airlift, traditional sparged-
agitated
designs, or in shaking culture.
The culture conditions employed, including temperature, pH, aeration rate,
agitation rate, culture duration, and the like, may be determined empirically
by
one of skill in the art to maximize L-sorbose and 2-keto-L-gulonic acid
production. The selection of specific culture conditions depends upon factors
such
as the particular inventive microorganism strain employed, medium composition
and type, culture technique, and similar considerations.
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Illustrative examples of suitable methods for recovering 2-KLG are
described in U.S. Pat. Nos. 5,474,924; 5,312,741; 4,960,695; 4,935,359;
4,877,735; 4,933,289; 4,892,823; 3,043,749; 3,912,592; 3,907,639 and
3,234,105.
According to one such method, the microorganisms are first removed
from the culture broth by known methods, such as centrifugation or filtration,
and
the resulting solution concentrated in vacuo. Crystalline 2-KGL is then
recovered
by filtration and, if desired, purified by recrystallization. Similarly, 2-KGL
can be
recovered using such known methods as the use of ion-exchange resins, solvent
extraction, precipitation, salting out and the like.
When 2-KGL is recovered as a free acid, it can be converted to a salt, as
desired, with sodium, potassium, calcium, ammonium or similar cations using
conventional methods. Alternatively, when 2-KGL is recovered as a salt, it can
be converted to its free form or to a different salt using conventional
methods.
All patents and publications referred to herein are expressly incorporated
by reference. Having now generally described the invention, the same will be
more readily understood through reference to the following Examples which are
provided by way of illustration, and are not intended to be limiting of the
present
invention, unless specified.
Examples
Example 1
Isolation and Characterization of SDHfrom G. suboxydans KCTC 2111
An improved method for the purification of the pyrroloquinoline quinone
(PQQ)-dependent SDH of the invention from G. suboxydans KCTC 2111 is
presented. Improvements over the original purification scheme (Choi, E. S. et
al.,
FEMSMicrobiol. Lett., 125:45 (1995)) relate to greater subunit resolution and
improved stability of enzyme activity.
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Step l:Cultivation of G. suboxydans KCTC 2111
G. suboxydans KCTC 2111 was inoculated into 5 ml of SYP medium (5%
D-sorbitol, 1 % BactoPeptone and 0.5% yeast extract) and incubated at
30°C for
20 hours. One milliliter (ml) of this culture were transferred to 50 ml of the
same
medium in a 500 ml flask and cultivated at 30°C for 20 hours on a
rotary shaker
(180 rpm). The culture thus prepared was used as an inoculum for a 5 L jar
fermentor containing 3 L of the same medium, and the 3 L culture was grown to
early stationary phase.
Step 2:Preparation of the Membrane Fraction
Cells were harvested by centrifugation at 12,000 g for 10 min, washed
once with 10 mM sodium acetate buffer (pH 5.0) and disrupted with glass beads
(0.1 mm in diameter) in a bead beater (Edmund Buhler, Vi 4) for 90 sec at
4°C.
The homogenate thus prepared was centriftiged at 12,000 g for 5 min to remove
cell debris and glass beads. The resulting supernatant was centrifuged at
100,000
g for 60 min, and a crude membrane fraction was obtained as a precipitate.
Step 3:Solubilization of SDH from the Membrane Fraction
The crude membrane fraction was resuspended at 40 mg of protein per ml
and solubilized with 1.5% n-octyl glucoside by stirring for 2 hours at
4°C. The
resultant suspension was centrifuged at 12,000 g for 10 min to give a
supernatant,
designated as the solubilized SDH fraction. All the solutions employed in the
purification procedures contained 0.75% n-octyl glucoside.
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Step 4:Enzyme Activity Assay
Enzyme activity was assayed spectrophotometrically using 2,6-
dichlorophenol indophenol (DCIP) as an artificial electron acceptor and
phenazine methosulphate (PMS) as an electron mediator. The reaction mixture
contained 50 mM sodium acetate buffer (pH 5.0), 10 mM MgCh, 5 mM CaCl2,
5 mM KCN, 0.1 mM PMS, 0.12 mM DCIP, 250 mM D-sorbitol, 0.75% n-octyl
glucoside and enzyme solution in a total volumn of 1.0 ml. The molar
extinction
coefficient of e= 5,600 cm' M-' for DCIP at pH 5.0 was employed. One unit of
enzyme activity was defined as the amount of enzyme catalyzing reduction of 1
qmol of DCIP per min.
Enzyme activity was determined also by the Ferric-Dupanol method
(Wood, W.A. et al., Meth. Enzymol. 5:287 ( 1962)) for the reconstituted
subunits
described in step 6 of Example 1. Subunit proteins were preincubated either
singly or in different combinations for 5 min at 25°C. The reaction was
started
by the addition of 10 mM (final concentration) of potassium ferricyanide and
250
mM (final concentration) D-sorbitol. After an appropriate time, the reaction
was
stopped by adding the ferric sulfate-Dupanol solution (Fez(S04)3~nH,O Sg/L,
Dupanol (sodium lauryl sulfate) 3g/L and 85% phosphoric acid 95m1/L), and the
absorbance of the Prussian color was determined at 660 nm in a
spectrophotometer.
Step S:Ion-exchange Chromatography
The solubilized fraction was loaded onto a CM-TSK 650 (S) (Merck)
column (2.5 x 20 cm) equilibrated with 10 mM sodium acetate (pH 5.0). The
CM-TSK column was eluted with a linear gradient (from 10 mM to 500 mM) of
sodium acetate. Active fractions were pooled and concentrated by
ultrafiltration
using a membrane filter (Amicon, YM 10) and loaded onto a DEAE-TSK 650 (S)
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(Merck) column (2.5 x 20 cm). The DEAE-TSK column was eluted isocratically
with a 10 mM sodium acetate buffer of either pH 5.0 or pH 6Ø
As shown in Figure 1, when eluted at pH 6.0 (Fig. 1-B), the resolution of
the activity peaks was much higher than with an elution at pH 5.0 (Fig.l-A),
and
the enzyme activity peaks I and II were completely separated. When the enzyme
activity of the fractions was measured immediately after the elution and one
hour
after the elution, it was found that the enzyme activity of the fractions in
peaks I
and II eluting together at pH 5.0 did not differ signficantly with time. In
the case
of the fractions in peaks I and II eluting separately at pH 6.0, however, the
enzyme activity in peak I decreased to less than one tenth of the original
value
within one hour after the elution, whereas the enzyme activity in the peak II
remained unchanged.
Fractions containing peaks I and II eluted at pH 6.0 on DEAE-TSK
column were analyzed by SDS-PAGE (Laemmli, U. K., Nature, 227, 680 ( 1970))
(Figure 2). Figure 2-A shows fractions of peak I. Column M shows standard
molecular weight markers. As shown in columns 1 through 4, the 75 kDa first
subunit band and the 50 kDa second subunit band could be observed but the 29
kDa third subunit band was not observed. In this case, one hour after elution,
the
enzyme activity was reduced by more than ten fold. Figure 2-B shows the
fractions of peak II. Column M shows standard molecular weight markers. As
shown in columns 5 through 8, it was observed that fractions of peak II
contained
the 29 kDa third subunit band in addition to the 75 kDa and 50 kDa subunits.
This observation indicated that the 29 kDa third subunit might play an
important
role in the stability of SDH.
Step 6: SDH Reconstitution with Different Subunits
Since the increase of the pH of the elution buffer from 5.0 to 6.0 during
DEAE column chromatography increased the resolution of the chromatographic
peaks, the pH of the elution buffer was further increased to pH 6.5. Partially
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purified SDH provided by CM-TSK 650 (S) column chromatography as described
in step 5 of Example 1 was loaded onto DEAE-TSK 650 (S) column (2.5 x 16.5
cm) pre-equilibrated with 20 mM sodium phosphate buffer (pH 6.5), and the
column was isocratically eluted with 180 ml of the same buffer followed by a
gradient elution with 160 ml each of 20 mM and 500 mM of sodium phosphate
buffer (pH 6.5). Column fractions were analyzed by SDS-PAGE.
As shown Figure 3, the first subunit of 75 kDa eluted first (peak I)
followed by the third subunit of 29 kDa (peak II) during the isocratic
elution, and
the second subunit of 50 kDa eluted later during the gradient elution (peak
III).
As shown in Figure 4, the SDS-PAGE analysis of the three peaks indicated that
the three subunits were essentially pure and completely separated from each
other.
Since the three subunits could be purified and separated into individual
species under non-denaturing conditions, reconstitution experiments were
conducted to determine the role of each subunit for catalytic activity of the
SDH
enzyme. Approximately 100 pmoles of each subunit obtained from the DEAE
column by elution at pH 6.5 were preincubated either singly or in different
combinations and assayed for the enzyme activity by Ferric-Dupanol method as
described in step 4 of Example 1 (Table 2). Each subunit alone showed a very
low level of activity. When the first and the second subunits were
reconstituted,
enzyme activity increased by two times compared with the first subunit alone.
When the third subunit was added to the first subunit, and to the mixture of
the
first and the second subunits, the enzyme activity increased by about 30 and
20
times, respectively, These results indicated that the third subunit plays an
important role for the catalytic activity of SDH as well as the stability of
the
enzyme.
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Table 2
Subunit (kDa) SDH enzyme activity (OD66o)
75 0.029
50 0.002
29 0.004
75 + 50 0.051
75 + 29 0.944
75 + 50 + 29 0.692
Example 2
Determination of the N terminal Amino Acid Sequences of the SHD Subunits
The purified SDH prepared in Example 1 was subjected to SDS-PAGE
( 12.5% gel), and the separated proteins were electroblotted onto a
polyvinylidene
difluoride (PVDF) membrane (Bollag, D. M. and Edelstein, S. J., Protein
Methods, Wiley-Liss, Inc., 8 (1991)). After visualization with ponceau S
stain,
the section of membrane containing each SDH subunit was cut into pieces, and
the membrane pieces for each subunit were applied directly to an amino acid
sequence analyzer (Applied Biosystems, Model 477A) for N-terminal amino acid
sequence analysis.
The resultant data for the first and the third subunits are shown in Table 3
(SEQ ID Nos:9 and 11 ). The N-terminal amino acid sequence of the second
subunit could not be obtained, most likely because of a blocked N-terminus. A
similar finding of a blocked N-terminus was reported for the cytochrome c
subunit of the alcohol dehydrogenase complex of another acetic acid bacterium,
Acetobacter pasteurianus (Takemura, H. et al., J. Bacteriol. 175, 21, 6857
(1993)), where the blockage of the N-terminus was ascribed to the modification
of glutamate residue at the N-terminus to a pyroglutamate residue, which is
recalcitrant to the Edman degradation during N-terminal amino acid sequencing.
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Therefore, in order to obtain N-terminal sequence for the second subunit
of purified SDH, the isolated SDH protein was first treated with pyroglutamate
aminopeptidase to release the potentially blocked N-terminus. Briefly, about
50
pg of purified SDH from Example 1 was dissolved in a digestion buffer ( 100 mM
sodium phosphate buffer, 10 mM EDTA, 5 mM dithiothreitol and 5% (v/v)
glycerol, pH 8.0) and incubated with 3.7~ ~g of pyroglutarnate aminopeptidase
(Boehringer Mannheim) at 4°C for 18 hours, followed by an additional
incubation
for 4 hours at 25°C. After incubation, the reaction mixture was
subjected to SDS-
PAGE, electroblotted onto a PVDF membrane, and the second subunit band on
the membrane was excised and analyzed for the N-terminal amino acid sequence
as described above. Ten residues of N-terminal amino acid sequence is shown
in Table 3 as Sequence ID. No:lO.
Table 3
Sequence ID N-terminal amino acid sequence
No.
SEQ ID N0:9 EDTGTAITNADQHPG
SEQ ID NO:10 DADDALIQRG
SEQ ID NO:I AGTPLKIGVSFQEMNNPYFVTMKDA
1
Example 3
Determination of Internal Amino Acid Sequence for the Third Subunit
For the third subunit, internal amino acid sequences were also determined
in addition to the N-terminal amino acid sequence for the facilitated cloning
of
the corresponding gene. About 7 ~g of the third subunit protein isolated as
described in step 6 of Example 1, was digested with trypsin (Boehringer
Mannheim) as previously described (Matsudaira, P.T., A Practical Guide to
Protein and Pept Purification for Microsequencing Academic Press p. 37
( 1989)). The tryptic digest was separated by HPLC using a Brownlee SPHERI-5
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RP 18 column (0.2 x 22 cm), and the column was eluted with a linear gradient
of
15-70% acetonitrile for 60 minutes at 210 microliter/minute. The elution was
monitored at 214 nm, and the well separated three peptide peaks were collected
(designated 1. 5 and 7 in Fig. 5) and analyzed for their amino acid sequence
as
described in (Example 2). The internal amino acid sequences for the peaks, 1,
5
and 7 are shown below in Table 4 (SEQ ID NO: 12, 13, and 14, respectively.
Table 4
Sequence ID No. Internal amino acid
sequence
SEQ ID N0:12 HSDIK
SEQ ID N0:13 NYDAGFK
SEQ ID N0:14 KWGAGVPK
Example 4
Primer Design and Isolation of a 1.53 kb DNA Fragment
Containing a Portion of the SDH Gene
Based on the N-terminal amino acid sequence of the first subunit (SEQ
ID N0:9), two degenerate primers, primer 1 (5' - CCGGAATTC GAA(G)
GAT(C) ACI GGI ACI GC-3') (SEQ ID NO:1 S) and primer 2 (5'-ATT(C,A) ACI
AAT(C) GCI GAT(C) CAAG) CAT(C) CC-3')(SEQ ID N0:16), were
synthesized.
The genomic DNA isolated from G. suboxydans KCTC 2111 using the
method of Takeda and Shimizu (Takeda and Shimizu, J. Ferm. Bioeng., 72:1
(1991)) was partially digested with BamHI. The plasmid pBlueseript SK
(Stratagene) was restricted with Nael and BamHI and the BamHI partial digest
of
genomic DNA was ligated to the BamHI site of the plasmid.
Thirty cycles of polymerase chain reaction (PCR) was performed in
accordance with the single specific primer PCR (SSP-PCR) method (White, B.,
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SSP-PCR and genome walking, in Method in Mol. Biol., PCR protocols, Humana
Press, 15:339 (1993)) to isolate a clone (#SDH2-1) 1.53 kb in size.
The ligation reaction mixture prepared above was amplified with a gene
specific primer, the primer 1, and the T7 primer of the vector. Although the
generic T7 primer anneals to the ends of all ligated fragments, the resulting
products increase only linearly. However, simultaneous annealing of the gene
specific primer 1 and T7 primer to the specific product results in exponential
amplification of the specific primary product. Secondary PCR carried out with
a nested primer, the gene specific primer 2, and the T7 primer generated a
specific
secondary product, which confirmed the specificity of the primary PCR.
The PCR product was ligated to plasmid pT7 Blue (Novagen) and
transformed into Escherichia coli DHSa by SEM protocol (moue, H. et al., Gene,
96:23 ( 1990)). Transformants were cultivated in an LB medium ( 1 % Bacto-
Tryptone, 0.5% yeast extract and 1 % NaCI) supplemented with 100 ~g/ml of
ampicillin. Subsequently, the plasmid was extracted by alkaline lysis method
(Sambrook, J. et al., Molecular Cloning, CSH Press, p. 125 (1988)).
PCR with primer 1 or primer 2 together with the T7 primer using this
plasmid as a template yielded a positive reaction. Partial nucleotide
sequencing
of #SDH2-1 fragment further confirmed that the derived amino acid sequence
downstream of the primer 1 binding site matched with the experimentally
determined N-terminal amino acid sequence. However, #SDH2-1 contained only
a part of the first subunit gene.
Example S
Isolation of Lambda GEM S-1 Clone Containing SDH Subunit Genes
Using the 1.53 kb DNA Fragment as a Probe
The #SDH2-1 isolated in Example 4 was labeled with DIG Labeling and
Detection Kit (The DIG System User's Guide for Filter Hybridization. p. 6-9,
Boehringer Mannheim (1993)).
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To construct a genomic DNA library of G. suboxydans KCTC 2111, the
genomic DNA isolated from G. .suboxydans KCTC 2111 using the method
described in Example 4 was partially digested with Sau3AI. Partially digested
DNA was electrophoresed in a 0.8% agarose gel and the DNA of 15 to 23 kb in
size was eluted using QLAEX II Gel Extraction Kit (QIAGEN). The eluted DNA
was then ligated into the BamHI site of Lambda GEM- 11 vector (Promega). The
ligation mixture was packaged into phage lambda particles using the Packagene
In Vitro Packaging System (Promega) according to the instruction manual. E.
coli LE392 cells were grown in TB medium ( 1 % Bacto-Tryptone and 0.5% NaCI)
supplemented with 0.2% maltose and 10 mM MgS04, at 30°C and stored at
4°C
when the ODboo had reached 0.6. The packaging mixture was added to the cell
suspension, and the mixture was incubated for 30 min at 37°C to allow
infection.
To this mixture, 3m1 of molten (45°C) TB top agar (0.8% Bacto-Agar
in TI3
medium containing 10 mM MgS04) was added, vortexed gently and immediately
poured onto LB plates. The plates were incubated inverted at 37°C
overnight.
The lambda phage plaques were immobilized on nylon membranes
(Amersham) and the membranes were prehybridized in a hybridization oven
(Hybaid) using a prehybridization solution (5X SSC, 1 % (w/v) blocking
reagent,
0.1% N-lauroylsarcosine, 0.02% SDS and 50% (v/v) formamide) at 42°C for
3
hours. Then the membranes were hybridized using a hybridization solution (DIG-
labeled #SDH2-1 probe diluted in the prehybridization solution) at 42°C
for 16
hours. Eleven plaques among about 20,000 plaques of the lambda phage gave
positive signals by plaque hybridization (The DIG System User's Guide for
Filter
Hybridization. Boehringer Mannheim (1993)) with the DIG-labeled #SDH2-1
probe.
The lambda DNAs were isolated from positive lambda clones and purified
with Lambda DNA Purification Kit (Stratagene). The isolated lambda DNAs
were digested with BamHl and subjected to a 0.7% agarose gel electrophoresis.
The DNA fragments separated on the gel were transferred onto a nylon membrane
and analyzed again by Southern hybridization with #SDH2-1 as probe under the
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same condition as described above. A clone which gave a positive signal was
selected, and the insert DNA of 15 kb was excised with XhoI from this clone
and
subsequently cloned into the Xhol site of pBluescript SK give Lambda GEM 5-1.
The Lambda GEM 5-1 clone was mapped by digestion with several different
restriction enzymes. Figure 6 presents the restriction enzyme map of Lambda
GEM 5-1.
Example 6
Determination of the Nucleotide Seguence of the 5.7 kb Pstl Fragment
in the Lambda GEM 5-1 Clone
The positive clone, Lambda GEM 5-1, obtained in Example 5 was
mapped with several different restriction enzymes and analyzed by Southern
hybridization as described in Example 5. A 5.7 kb Pstl fragment hybridizing
with
#SDH2-1 was subcloned, and the nucleotide sequence was determined. To make
the DNA sequencing simpler, three overlapping subclones, S1, S2 and S3, were
constructed using restriction enzyme sets, KpnI-Pstl, Notl-Sacll and Pstl-
SacI,
respectively, as shown in Figure 7. A set of deletion clones was prepared for
each
subcIone using Exo III-Mungbean Deletion Kit (Stratagene). The nucleotide
sequencing reaction was done by Dye Terminator Cycle Sequencing Ready
Reaction Kit (Applied Biosystems), and the sequence was determined in an
automatic DNA sequencer (Applied Biosystems, Model 373A).
Figure 8 shows the nucleotide sequence of 4,830 by in the 5.7 kb Pstl
fragment (SEQ ID N0:7). The sequenced DNA contains two open reading
frames (ORFs) of 2,265 and 1,437 nucleotides. The first ORF encodes the first
subunit. The first subunit gene is preceded by a Shine-Dalgarno sequence,
"AGGA" positioned at 651-654 bp. The 34 amino acid signal sequence of the
first subunit is positioned at 665-766 by of SEQ ID N0:7. The coding sequence
of the mature part of the first subunit protein is positioned at 767-2,929 by
of
SEQ ID N0:7, which encodes a 720 amino acid polypeptide whose derived N-
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terminal amino acid sequence is in perfect agreement with 15 amino acid
residues
obtained by N-terminal amino sequence analysis.
The first ORF was followed by the second ORF, the two ORF's being
interrupted by a short intergenic region. The second ORF encodes the second
subunit. The Shine-Dalgarno sequence (AGGA) of the second subunit gene is
found at 2,950-2,953 by SEQ ID N0:8. and the structural gene is positioned at
2,964-4,400 by of SEQ ID N0:8. The second subunit gene encodes a 478 amino
acid polypeptide, including a signal sequence of 36 amino acids. The derived
amino acid sequence of the mature polypeptide showed a perfect match with the
experimentally obtained sequence for the sample treated with pyroglutamate
aminopeptidase. Downstream of the stop codon of the second subunit gene,
inverted repeat sequences are found.
The calculated molecular weights of the mature proteins of the first and
the second subunit are 79 kDa and 48 kDa, respectively, which are in good
agreement with the experimental values of 75 kDa and 50 kDa, respectively, as
determined by SDS-PAGE.
In addition it was also found that signature PQQ-binding sequences,
consensus sequences appearing characteristically in the amino- and carboxy-
terminals of PQQ-dependent dehydrogenases, are present in the first subunit
gene.
In Table 5, the signature PQQ-binding sequence in amino-terminal part is shown
as Sequence ID N0:17, and the signature sequence existing in the carboxy-
terminal part is shown as Sequence ID No:l8 (Here, X represents an arbitrary
amino acid.). The amino-terminal signature sequence occurs at position 812-898
bp, and the carboxy-terminal signature sequence occurs at position 1,490-1,555
bp. These data provide additional evidence that the first subunit contains
pyrroloquinoline quinone (PQQ) as cofactor.
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Table 5
[SEQ ID NO: 17]
[D/E/N]-W-X-X-X-G-[R/K]-X-X-X-X-X-X-[F/Y/W]-S-X-X-X-X-[L/I/V/M]-X-X-
X-N-X-X-X-L-[R/K]
( [SEQ ID NO: 18]
W-X-X-X-X-Y-D-X-X-X-[D/N]-[L/I/V/M/F/Y)- [L/I/V/M/F/Y]-[ L/I/V/M/F/Y]-
[L/I/V/M/F/Y]-X-X-G-X-X-[S/T/A)-P
In addition, it was also discovered that a single heme-binding sequence
occured at position 2,612-2,626 by in the first subunit gene (SEQ ID N0:19 in
Table 6 below), and three heme-binding sequence occurred at the following
positions: 3,129-3,143; 3,573-3,587 and 3,981-3,995 by in the second subunit
gene (Here, XA represents an arbitrary amino acid. X$, is an arbitrary amino
acid
different from XA.).
Table 6
[SEQ iD N0:19]
C-XA - XB -C-H
A database search for homologous sequences determined that the DNA
sequence of the first subunit gene showed a great degree of similarity to many
dehydrogenases containing pyrroloquinoline quinone (PQQ) as cofactor: In
particular, the alcohol dehydrogenases ofAcetobacter polyoxogenes (Tamaki, T.
et al., Biochim. Biophys. Acta, 1088, 292 (1991)) and Acetobacter aceti (moue,
T. et al., J. Bacteriol.,171, 3115 ( 1989)) are 77% and 70% identical,
respectively,
to the first subunit gene. A search of the database with the second subunit
gene
provided the greatest degree of similarity, matching the cytochrome c of G.
suboxydans IFO 12528 (Takeda and Shimizu, J. Ferm. Bioeng., 72:1 ( 1991 ))
with
a nucleotide sequence identity of 83% and an amino acid sequence identity of
88%.
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Example 7
Primer Design and PCR Cloning of 320 by DNA Fragment
Containing a Portion of the Third Subunit Gene
The nucleotide sequence analysis of the first and the second subunit genes
described in Example 6 indicated that the isolated operon clone did not
contain
the third subunit gene. Further sequencing of the 5' and 3' flanking regions
also
failed to show the presence of the third subunit. Therefore, in order to
isolate the
gene encoding the third subunit, degenerate primers were synthesize based on
the
amino acid sequence information. in order to generate by PCR a short DNA
fragment containing a portion of third subunit gene for use as probe.
Based on the N-terminal amino acid sequence (SEQ ID NO:11) of the
mature third subunit protein obtained in Example 2 and one of the internal
amino
acid sequence (SEQ ID N0:13) of the tryptic peptides obtained in Example 3,
two
degenerate primers, primer 3 (5'-GGGAATTC TTT(C) CAA(G) GAA(G) ATG
AAT(C) AA-3') (SEQ ID N0:20) and primer 4(5'-GGGAATTC TT GAA
A(G)CC NGC A(G)TC A(G)TA-3')(SEQ ID N0:21 ), respectively, were
synthesized.
A PCR reaction was done with primers 3 and 4 using genomic DNA of
G. suboxydans KCTC 2111 prepared by the method of Takeda and Shimizu
(Takeda and Shimizu, J. Ferm. Bioeng. , 72 1 ( 1991 )) as template. The
reaction
generated a 320 by DNA fragement. This 320 by PCR product was ligated to
pBluescript SK (Stratagene) and transformed into E. coli DHSa by the SEM
protocol (moue, H. et al., Gene, 96, 23 (1990)). Transformants were cultivated
in an LB medium supplemented with 100 ~ g/ml of ampicillin. Subsequently, the
plasmid was extracted by the alkaline lysis method (Sambrook, J. et al.,
Molecular Cloning, CSH Press, (1988)). Subcloning was verified by performing
a PCR reaction with primer 3 and primer 4 using this plasmid DNA as a
template.
Partial nucleotide sequencing of the 320 by fragment further confirmed that
the
derived amino acid sequence do~mstream of the primer 3 binding site matched
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with the experimentally determined N-terminal amino acid sequence. However,
the 320 by fragment contained only the N-terminal part of the third subunit
gene.
Example 8
Isolation of the Third Subunit Gene Using 320 by DNA Fragment as Probe
The 320 by DNA fragment isolated in (Example 7) was labeled with DIG
Labeling and Detection Kit (The DIG System User's Guide for Filter
Hybridzation. p6-9, Boehringer Mannheim (1993)). The genomic DNA isolated
from G. suboxydans KCTC 2111 using the method of Takeda and Shimizu
(Takeda and Shimizu, J. Ferm. Bioeng. , 72, ( ( 1991 )) was digested with
BamHI,
CIaI, EcoRI, HindIII, PstI, or XhoI, electrophoresed in a 0.8% agarose gel and
transferred to a nylon membrane (NYTRAN, Schleicher & Schuell) as described
(Southern, E.M., J. Mol. Biol. 98, 503 (1975)). The membrane was prehybridized
in a hybridization oven (Hybaid) using a prehybridization solution (5X SSC, 1%
(w/v) blocking reagent, 0.1% N-lauroylsarcosine, 0.2% SDS and 50% (v/v)
formamide) at 42 ° C for 2 hours. Then the membrane was hybridized
using a
hybridization solution (DIG-labeled probe diluted in the prehybridization
solution) at 42°C for 12 hours. Southern hybridization gave a strong
discrete
signal for each enzyme used. DNA corresponding to the positive signal at 4.5
kb
CIaI was eluted and cloned into pBluescript SK to construct a mini-library.
The
mini-library was screened for the positive clone by repeating the Southern
hybridization as described above. A clone which gave a positive signal was
selected and designated ClaI-#69. Figure 9 presents the restriction enzyme map
of CIaI-#69.
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Example 9
Nucleotide Sequence Analysis of the 4.5 kb Clal Fragment
Containing tire Third Subunit
The nucleotide sequence of the third subunit gene in the 4.5 kb CIaI-#69
clone was determined and analyzed. To facilitate the DNA sequencing, several
overlapping restriction fragments were subcloned, and the nucleotide sequence
of each clone determined. The nucleotide sequencing reaction was done by Dye
Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and the
sequence was determined in an automatic DNA sequencer (Applied Biosystems,
Mode1373A).
Figure 10 shows the nucleotide sequence of 2700 by of the 4.5 kb CIaI
fragment (SEQ ID N0:8). The sequenced DNA contains an open reading frame
(ORF) of 921 nucleotides, which encodes the third subunit polypeptide. The
third
subunit gene is preceded by a potential Shine-Dalgarno sequence (AGG)
positioned at 1,375-1,377 bp. The amino acid signal sequence ofthe third
subunit
polypeptide is positioned at 1,384-1,461 bp. The coding sequence of the mature
part of the third subunit protein is positioned at 1,462-2,304 bp, encoding a
280
amino acid polypeptide whose derived N-terminal amino acid sequence was in
perfect agreement with the 25 amino acid residues obtained by N-terminal amino
sequence analysis. The calculated molecular weight of the mature protein of
the
third subunit is 29,552 Da, which was in good agreement with the experimental
value of 29 kDa determined by SDS-PAGE.
